Cancer often develops through the disruption of proper responses to extracellular signals. Thus, understanding how normal cells sense and generate proper responses to changes in environment is important. Normally, transduction of extracellular signals into cellular responses involves a cascade of biochemical events that eventually induce gene expression. These events are mediated by transcription factors (TF), a class of DNA-binding proteins which dictate the nature of genes expressed. Deregulation of TF activities can lead to amplification of anomalies via uncontrolled gene expression. Activation of a critical TF, NF-kappaB, by extracellular signals normally occurs only transiently since NF-kappaB activates synthesis of its own inhibitor, IkappaBalpha. By contrast, deregulation of NF-kappaB activity, frequently seen in human cancers, must counteract this feedback mechanism to maintain constitutive (constant) NF-kappaB activation. How and why is NF-kappaB anomalously activated? Research in this laboratory has demonstrated that murine B cells, a rare example with non-pathological constitutive NF- kappaB activation, maintain such activity by degrading newly synthesized IkappaBalpha via a previously uncharacterized mechanism. Our preliminary data also suggest that certain human breast cancer cells aberrantly activate NF-kappaB by utilizing this alternative mechanism. Thus, the proposed research will test the hypothesis that a novel IkappaBalpha degradation mechanism is required for constitutive NF- kappaB activation in B cells and certain human breast cancer cells. In aim 1, mutational analysis will be employed to identify IkappaBalpha sequences that specify degradation by this novel pathway. Under Aim 2, the functional role of this alternative pathway will be determined in both induction and maintenance phases of constitutive NF-kappaB activation in B cells, by further specifying how proteasome-dependent NF-kappaB activation becomes proteasome-independent via a switch in IkappaBalpha degradation mechanisms. Aim 3 will test the mechanism and role of constitutive NF-kappaB activation in breast cancer cells by focusing on the alternative IkappaBalpha degradation in relation to cell survival function. This research program will provide not only a mechanistic insight into how an alternative pathway restricted to normal B cell function may be abnormally employed by cancer cells by also molecular reagent tools to probe other constitutive NF-kappaB activation systems.